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https://www.readbyqxmd.com/read/27681498/development-of-a-microflow-system-for-in-cell-footprinting-coupled-with-mass-spectrometry
#1
Aimee Rinas, Vishaal S Mali, Jessica A Espino, Lisa M Jones
Fast photochemical oxidation of proteins (FPOP) has become a valuable tool for protein structural characterization. The method has recently been demonstrated to oxidatively modify solvent-accessible sites of proteins inside live cells (IC-FPOP). However, the flow system used for in vitro analysis is not well-suited for IC-FPOP as a number of factors can lead to cell aggregation, causing inconsistent labeling and clogging. Here, we present an IC-FPOP flow system that centrally focuses the cells, ensuring consistent radiation exposure...
October 7, 2016: Analytical Chemistry
https://www.readbyqxmd.com/read/27568528/conformational-sensitive-fast-photochemical-oxidation-of-proteins-and-mass-spectrometry-characterize-amyloid-beta-1-42-aggregation
#2
Ke Sherry Li, Don L Rempel, Michael L Gross
Preventing and treating Alzheimer's disease require understanding the aggregation of amyloid beta 1-42 (Aβ1-42) to give oligomers, protofibrils, and fibrils. Here we describe footprinting of Aβ1-42 by hydroxyl radical-based fast photochemical oxidation of proteins (FPOP) and mass spectrometry (MS) to monitor the time-course of Aβ1-42 aggregation. We resolved five distinct stages characterized by two sigmoidal behaviors, showing the time-dependent transitions of monomers-paranuclei-protofibrils-fibrillar aggregates...
September 21, 2016: Journal of the American Chemical Society
https://www.readbyqxmd.com/read/27500903/fast-photochemical-oxidation-of-proteins-maps-the-topology-of-intrinsic-membrane-proteins-light-harvesting-complex-2-in-a-nanodisc
#3
Yue Lu, Hao Zhang, Dariusz M Niedzwiedzki, Jing Jiang, Robert E Blankenship, Michael L Gross
Although membrane proteins are crucial participants in photosynthesis and other biological processes, many lack high-resolution structures. Prior to achieving a high-resolution structure, we are investigating whether MS-based footprinting can provide coarse-grained protein structure by following structural changes that occur upon ligand binding, pH change, and membrane binding. Our platform probes topology and conformation of membrane proteins by combining MS-based footprinting, specifically fast photochemical oxidation of proteins (FPOP), and lipid Nanodiscs, which are more similar to the native membrane environment than are the widely used detergent micelles...
September 6, 2016: Analytical Chemistry
https://www.readbyqxmd.com/read/27343183/fpop-lc-ms-ms-suggests-differences-in-interaction-sites-of-amphipols-and-detergents-with-outer-membrane-proteins
#4
Thomas G Watkinson, Antonio N Calabrese, James R Ault, Sheena E Radford, Alison E Ashcroft
Amphipols are a class of novel surfactants that are capable of stabilizing the native state of membrane proteins. They have been shown to be highly effective, in some cases more so than detergent micelles, at maintaining the structural integrity of membrane proteins in solution, and have shown promise as vehicles for delivering native membrane proteins into the gas phase for structural interrogation. Here, we use fast photochemical oxidation of proteins (FPOP), which irreversibly labels the side chains of solvent-accessible residues with hydroxyl radicals generated by laser photolysis of hydrogen peroxide, to compare the solvent accessibility of the outer membrane protein OmpT when solubilized with the amphipol A8-35 or with n-dodecyl-β-maltoside (DDM) detergent micelles...
June 24, 2016: Journal of the American Society for Mass Spectrometry
https://www.readbyqxmd.com/read/27067899/probing-the-time-scale-of-fpop-fast-photochemical-oxidation-of-proteins-radical-reactions-extend-over-tens-of-milliseconds
#5
Siavash Vahidi, Lars Konermann
Hydroxyl radical (⋅OH) labeling with mass spectrometry detection reports on protein conformations and interactions. Fast photochemical oxidation of proteins (FPOP) involves ⋅OH production via H2O2 photolysis by UV laser pulses inside a flow tube. The experiments are conducted in the presence of a scavenger (usually glutamine) that shortens the ⋅OH lifetime. The literature claims that FPOP takes place within 1 μs. This ultrafast time scale implies that FPOP should be immune to labeling-induced artifacts that may be encountered with other techniques...
July 2016: Journal of the American Society for Mass Spectrometry
https://www.readbyqxmd.com/read/26865209/assessment-of-pelvic-organ-prolapse-a-review
#6
K L Shek, H P Dietz
Female Pelvic organ prolapse (FPOP) is a highly prevalent condition. Its incidence is likely to increase further because of aging populations. There is a need to understand treatment and prevention better, and proper diagnosis is the first step towards these ends. Most importantly, we need to define what is 'normal' and 'abnormal'. In this review article we will focus on the clinical and ultrasound assessment of FPOP. Current data suggest pelvic floor injury is important in the pathogenesis of FPOP and as a risk factor for prolapse recurrence after surgery...
February 11, 2016: Ultrasound in Obstetrics & Gynecology
https://www.readbyqxmd.com/read/26679355/protein-footprinting-by-carbenes-on-a-fast-photochemical-oxidation-of-proteins-fpop-platform
#7
Bojie Zhang, Don L Rempel, Michael L Gross
Protein footprinting combined with mass spectrometry provides a method to study protein structures and interactions. To improve further current protein footprinting methods, we adapted the fast photochemical oxidation of proteins (FPOP) platform to utilize carbenes as the footprinting reagent. A Nd-YAG laser provides 355 nm laser for carbene generation in situ from photoleucine as the carbene precursor in a flow system with calmodulin as the test protein. Reversed-phase liquid chromatography coupled with mass spectrometry is appropriate to analyze the modifications produced in this footprinting...
March 2016: Journal of the American Society for Mass Spectrometry
https://www.readbyqxmd.com/read/26616261/is-it-necessary-to-diagnose-levator-avulsion-on-pelvic-floor-muscle-contraction
#8
H P Dietz, A Pattillo Garnham, R Guzmán Rojas
INTRODUCTION: Avulsion of the levator ani commonly occurs at vaginal birth. This condition is frequently diagnosed by translabial ultrasound (TLUS) during pelvic floor muscle contraction (PFMC). Some patients are unable to achieve a satisfactory PFMC, and in these cases avulsion is assessed at rest. The aim of this study was to validate this practice. METHODS: This is a retrospective study on 233 patients seen at a tertiary urogynecological centre. All underwent 4D TLUS, supine and after voiding...
November 30, 2015: Ultrasound in Obstetrics & Gynecology
https://www.readbyqxmd.com/read/26606592/a-dynamic-model-of-ph-induced-protein-g-e-higher-order-structure-changes-derived-from-mass-spectrometric-analyses
#9
Yelena Yefremova, Mahmoud Al-Majdoub, Kwabena F M Opuni, Cornelia Koy, Yuetian Yan, Michael L Gross, Michael O Glocker
To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino acid residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to acidic, whereas its secondary structure features remained nearly invariable. Limited proteolysis and surface-topology mapping of protein G'e by fast photochemical oxidation of proteins (FPOP) under neutral and acidic conditions reveal areas where higher order conformational changes occur on the amino-acid residue level...
January 5, 2016: Analytical Chemistry
https://www.readbyqxmd.com/read/26590487/pelvic-organ-prolapse-a-review
#10
Hans Peter Dietz
BACKGROUND: Female pelvic floor dysfunction encompasses a number of prevalent clinical conditions including urinary and faecal incontinence, obstructed defaecation, sexual dysfunction and female pelvic organ prolapse (FPOP). The latter is the most common condition and most likely to require surgical treatment. Neither aetiology nor pathophysiology of FPOP is fully understood. OBJECTIVE: This review will focus on the diagnosis and management of FPOP in primary care, but will also refer to recent research into aetiology, diagnosis, management and prevention of this condition...
July 2015: Australian Family Physician
https://www.readbyqxmd.com/read/26523679/determination-of-protein-folding-intermediate-structures-consistent-with-data-from-oxidative-footprinting-mass-spectrometry
#11
Florian Heinkel, Jörg Gsponer
The mapping of folding landscapes remains an important challenge in protein chemistry. Pulsed oxidative labeling of exposed residues and their detection via mass spectrometry provide new means of taking time-resolved "snapshots" of the structural changes that occur during protein folding. However, such experiments have been so far only interpreted qualitatively. Here, we report the detailed structural interpretation of mass spectrometry data from fast photochemical oxidation of proteins (FPOP) experiments at atomic resolution in a biased molecular dynamics approach...
January 29, 2016: Journal of Molecular Biology
https://www.readbyqxmd.com/read/26455423/hydroxyl-radical-dosimetry-for-high-flux-hydroxyl-radical-protein-footprinting-applications-using-a-simple-optical-detection-method
#12
Boer Xie, Joshua S Sharp
Hydroxyl radical protein footprinting (HRPF) by fast photochemical oxidation of proteins (FPOP) is a powerful benchtop tool used to probe protein structure, interactions, and conformational changes in solution. However, the reproducibility of all HRPF techniques is limited by the ability to deliver a defined concentration of hydroxyl radicals to the protein. This ability is impacted by both the amount of radical generated and the presence of radical scavengers in solution. In order to compare HRPF data from sample to sample, a hydroxyl radical dosimeter is needed that can measure the effective concentration of radical that is delivered to the protein, after accounting for both differences in hydroxyl radical generation and nonanalyte radical consumption...
November 3, 2015: Analytical Chemistry
https://www.readbyqxmd.com/read/26289617/warping-of-the-levator-hiatus-how-significant-is-it
#13
H P Dietz, M Severino, I Kamisan Atan, K L Shek, R Guzman Rojas
OBJECTIVES: The levator hiatus is the largest potential hernial portal in the human body. Excessive distensibility is associated with female pelvic organ prolapse (POP). Distension occurs not just laterally but also caudally, resulting in perineal descent and hiatal deformation or 'warping'. The aim of this study was to quantify the warping effect in symptomatic women, to validate the depth of the rendered volume used for the 'simplified method' of measuring hiatal dimensions and to determine predictors for the degree of warping...
August 2016: Ultrasound in Obstetrics & Gynecology
https://www.readbyqxmd.com/read/26249237/the-association-between-different-measures-of-pelvic-floor-muscle-function-and-female-pelvic-organ-prolapse
#14
Sissel H Oversand, Ixora Kamisan Atan, Ka Lai Shek, Hans Peter Dietz
INTRODUCTION AND HYPOTHESIS: We aimed to compare palpatory and translabial ultrasound (TLUS) measurements of pelvic floor muscle (PFM) function with symptoms and signs of female pelvic organ prolapse (FPOP) to determine a possible association. METHODS: We analysed data from 726 women with a mean age of 56 (SD 13.7, range 18-88) years, seen for symptoms of pelvic floor dysfunction between August 2011 and April 2013. The examination included a standardised interview and clinical assessment of FPOP with Pelvic Organ Prolapse Quantification (POP-Q) measurements, Modified Oxford Scale (MOS) grading and 4D TLUS...
December 2015: International Urogynecology Journal
https://www.readbyqxmd.com/read/26236917/development-of-structural-marker-peptides-for-cystic-fibrosis-transmembrane-conductance-regulator-in-cell-plasma-membrane-by-reversed-footprinting-mass-spectrometry
#15
Vahid Farrokhi, Bekim Bajrami, Reza Nemati, Adam J McShane, Franz Rueckert, Barrett Wells, Xudong Yao
A targeted mass spectrometry-based method is presented that adopts the fast photochemical oxidation of proteins (FPOP) for footprinting of cystic fibrosis transmembrane conductance regulator (CFTR) membrane transporter at its original plasma membrane location. Two analytical imperatives were sought: (1) overall simplification in data acquisition and analysis and (2) lower quantitation limits, which enabled direct analysis of intrinsically low-abundance transmembrane proteins. These goals were achieved by using a reversed-footprinting technique that monitored the unoxidized peptides remaining after the FPOP treatment...
September 1, 2015: Analytical Chemistry
https://www.readbyqxmd.com/read/25746386/using-hydroxyl-radical-footprinting-to-explore-the-free-energy-landscape-of-protein-folding
#16
Antonio N Calabrese, James R Ault, Sheena E Radford, Alison E Ashcroft
Characterisation of the conformational states adopted during protein folding, including globally unfolded/disordered structures and partially folded intermediate species, is vital to gain fundamental insights into how a protein folds. In this work we employ fast photochemical oxidation of proteins (FPOP) to map the structural changes that occur in the folding of the four-helical bacterial immunity protein, Im7. Oxidative footprinting coupled with mass spectrometry (MS) is used to probe changes in the solvent accessibility of amino acid side-chains concurrent with the folding process, by quantifying the degree of oxidation experienced by the wild-type protein relative to a kinetically trapped, three-helical folding intermediate and an unfolded variant that lacks secondary structure...
November 1, 2015: Methods: a Companion to Methods in Enzymology
https://www.readbyqxmd.com/read/25712620/dosimetry-determines-the-initial-oh-radical-concentration-in-fast-photochemical-oxidation-of-proteins-fpop
#17
COMPARATIVE STUDY
Ben Niu, Hao Zhang, Daryl Giblin, Don L Rempel, Michael L Gross
Fast photochemical oxidation of proteins (FPOP) employs laser photolysis of hydrogen peroxide to give OH radicals that label amino acid side-chains of proteins on the microsecond time scale. A method for quantitation of hydroxyl radicals after laser photolysis is of importance to FPOP because it establishes a means to adjust the yield of •OH, offers the opportunity of tunable modifications, and provides a basis for kinetic measurements. The initial concentration of OH radicals has yet to be measured experimentally...
May 2015: Journal of the American Society for Mass Spectrometry
https://www.readbyqxmd.com/read/25519854/an-improved-fast-photochemical-oxidation-of-proteins-fpop-platform-for-protein-therapeutics
#18
Ying Zhang, Don L Rempel, Hao Zhang, Michael L Gross
Unlike small-molecule drugs, the size and dynamics of protein therapeutics challenge existing methods for assessing their high order structures (HOS). To extend fast photochemical oxidation of proteins (FPOP) to protein therapeutics, we modified its platform by introducing a mixing step prior to laser irradiation to minimize unwanted H(2)O(2)-induced oxidation. This improvement plus standardizing each step yield better reproducibility as determined by a fitting process whereby we used a non-FPOP spectrum as a template to report the unmodified level...
March 2015: Journal of the American Society for Mass Spectrometry
https://www.readbyqxmd.com/read/25267085/fast-photochemical-oxidation-of-proteins-fpop-maps-the-epitope-of-egfr-binding-to-adnectin
#19
Yuetian Yan, Guodong Chen, Hui Wei, Richard Y-C Huang, Jingjie Mo, Don L Rempel, Adrienne A Tymiak, Michael L Gross
Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin ((10)Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose...
December 2014: Journal of the American Society for Mass Spectrometry
https://www.readbyqxmd.com/read/24927585/probing-the-paramyxovirus-fusion-f-protein-refolding-event-from-pre-to-postfusion-by-oxidative-footprinting
#20
Taylor A Poor, Lisa M Jones, Amika Sood, George P Leser, Manolo D Plasencia, Don L Rempel, Theodore S Jardetzky, Robert J Woods, Michael L Gross, Robert A Lamb
To infect a cell, the Paramyxoviridae family of enveloped viruses relies on the coordinated action of a receptor-binding protein (variably HN, H, or G) and a more conserved metastable fusion protein (F) to effect membrane fusion and allow genomic transfer. Upon receptor binding, HN (H or G) triggers F to undergo an extensive refolding event to form a stable postfusion state. Little is known about the intermediate states of the F refolding process. Here, a soluble form of parainfluenza virus 5 F was triggered to refold using temperature and was footprinted along the refolding pathway using fast photochemical oxidation of proteins (FPOP)...
June 24, 2014: Proceedings of the National Academy of Sciences of the United States of America
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